Isolation of pure and functional cell populations of a desired cell type is a prerequisite in a variety of therapeutic, diagnostic, and biotechnological applications.
Bonnafous et al., J. Immunol. Methods. 1983 Mar. 11; 58 (1-2):93-107 describe a cell affinity chromatography with ligands immobilized through cleavable mercury-sulfur bonds, that means ligands that are immobilized via covalent bonds. In this method, Bonnafous et al conjugate the organomercurial mersalyl to TRISACRYL® (Pall Corporation) beads bearing primary amino groups. According to Bonnafous et al, thiolated ligands can be covalently immobilized on this matrix through cleavable Hg—S bonds. Two model studies of cell separation are reported by Bonnafous et al: (i) concanavalin A thiolated with N-succinimidyl-3-(2-pyridyldithio)-propionate and immobilized on mersalyl-TRISACRYL®; mouse thymocytes bound to Con A-mersalyl-TRISACRYL® were eluted from the support by short thiol treatment which preserved cell viability; (ii) anti-dinitrophenyl antibodies modified with S-acetyl-mercaptosuccinic anhydride and immobilized on mersalyl-TRISACRYL®; sheep erythrocytes, previously labelled with trinitrobenzene sulfonic acid, bound to this support and were recovered by thiol treatment without hemolysis.
In this context it is noted that chromatography is a well-established technique for the separation of low molecular weight and high molecular weight molecules, including proteins. This technique has also been applied to cell separation, in particular in the form of affinity chromatography using immobilized ligands specific to a desired cell type, such as immunoligands. As an example, different T cell subsets have been separated by labelling with monoclonal immunoglobulins and loading onto a column with polyacrylamide beads, to which rabbit anti-mouse IgG was covalently bound (Braun, R., et al., Journal of Immunological Methods (1982) 54, 251-258). As a further example, lectin-affinity column chromatography, using Sepharose 6 MB covalently conjugated to Dolichos biflorus agglutinin, has been used to separate leukemic cells from healthy leukocytes (Ohba, H., et al, Cancer Letters (2002) 184, 207-214).
As cells are generally by magnitudes larger than proteins they hardly enter, in contrast to proteins, the pores of the beads of conventional chromatography sorbents. Using sorbents with large pores does not significantly overcome this separation phenomenon due to diffusional limitations. On the other hand, the surface area within pores only accessible for proteins usually largely exceeds the surface area accessible for both proteins and cells. Therefore, the use of conventional chromatography sorbents for the immobilization of proteinaceous or other receptor binding ligands for the generation of an affinity matrix for cells usually requires the use of a wasteful large excess of receptor binding ligands as most of them are immobilized in pores or cavities that cannot be accessed by the cells. Specific receptor binding reagents are often expensive and difficult to be produced at the desired scales thereby bringing this aspect to serious consideration. The use of monolithic sorbents in the form of cryogels has therefore been suggested as an alternative technique in affinity chromatography of cells (see e.g. Dainiak, M. B., et al., Adv. Biochem. Engin./Biotechnol. (2007), 106, 101-127). However, monolithic sorbents are scarce so that a desired sorbent may not be commercially available in the form of a monolithic column. Furthermore, in case of affinity chromatography, generally the need remains to remove a competing compound used to elute the desired cells from these cells. Potential advantages of monolithic sorbents in terms of cell viability may thus be reversed by additional procedures required to remove the compound used to elute the cells from the affinity chromatography column.
The most important currently used cell isolation methods are magnet-assisted cell sorting (MACS®, Miltenyi Biotec GmbH) and fluorescence-assisted cell sorting (FACS®, Becton Dickinson). Cell sorting by flow cytometry, where typically fluorophores, coupled to antibodies, are used to label cells, analyses cells individually. Cells are separated at high speed under very high pressures using a cell sorting apparatus. FACS® technology enables isolation of cells defined by a set of markers in one step by applying a corresponding set of antibodies with different fluorophores. The method is thus reliable, but time and cost intensive and laborious. Especially for the selection out of very large, diverse cell populations e.g., apheresis products containing 1×1010 cells very long sorting times of flow cytometers are unacceptable for an appropriate selection process. Another drawback of FACS® is that complex and interference-prone flow cytometers can hardly be adapted to a GMP environment necessary for isolating therapeutic cell products. Moreover, the applied pressures during the cell selection procedure may compromise cell effector function.
Magnet-assisted isolation of cells is a widely used system for research and therapeutic application. Although yield and purity of isolated cells are moderate compared to the FACS® technology the selection procedure is robust and does not require sophisticated automatization. The major drawbacks of the magnet-assisted isolation are the remaining staining reagents including the magnetic beads on the isolated cells which may compromise effector function of isolated cell populations. In addition no serial positive selection processes are possible due to these remaining magnetic reagents on the isolated cells. Serial positive selection procedures are mandatory for selecting cell populations defined by a set of markers. While still making use of a magnetic or fluorescent label, a significant advancement in the isolation of cells is the “Streptamer® technology that is, for example, described in International Patent Application WO 02/054065 and U.S. Pat. No. 7,776,562 and in which a receptor binding reagent exhibiting a low affinity binding to a receptor located on a surface of cell is used for the reversible staining and isolation of cells. In contrast to the currently used single positive selection combined with magnetic negative selection (aiming at removal of all cell populations but the one of interest) serial positive selection using the Streptamer® technology with removal of the low affinity receptor binding reagent after each selection generate cell populations of very high purity and yield.
It is an object of the present invention to provide a method and also an apparatus that overcomes the drawbacks of the known technology for isolation of cells, for example, FACS® and MACS® technology as described. For example, the present invention aims to provide a rapid, efficient and gentle cell selection procedure especially enabling serial positive cell selections for isolating complex cell populations such as regulatory T cells or central memory T-cells for research, diagnostic and especially therapeutic purposes. Ideally, this new method and apparatus should also be suitable for isolation of other complex biological materials than cells.
This object is solved by the subject matter of the independent claims, inter alia the methods, uses, and arrangements as recited in the independent claims.